Synthesis of Two-Photon Active Tricomponent Fluorescent Probe for Distinguishment of Biotin Receptor Positive and Negative Cells and Imaging 3D-Spheroid

Article abstract of DOI:10.1021/acs.orglett.8b02748

A fluorescence microscopy-based distinguishment between biotin receptor (BiR) positive and negative cell lines via receptor-mediated endocytosis has been demonstrated. A water-soluble, three-component, two-photon (2P) active solvatofluorochromic probe has been designed and synthesized. The applicability of the probe for 2P microscopy and 3D-spheroid was also assessed.

Full text of DOI:10.1021/acs.orglett.8b02748

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of Two-Photon Active Tricomponent Fluorescent Probe
for Distinguishment of Biotin Receptor Positive and Negative Cells
and Imaging 3D-Spheroid
Kaushik Pal,† Aman Sharma,‡ and Apurba L. Koner*,†
†Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri,
Bhopal-462066, India
‡ExoCan Healthcare Technologies Pvt. Ltd., Pune-411008, India
S
* Supporting Information
ABSTRACT: A fluorescence microscopy-based distinguish-
ment between biotin receptor (BiR) positive and negative cell
lines via receptor-mediated endocytosis has been demon-
strated. A water-soluble, three-component, two-photon (2P)
active solvatofluorochromic probe has been designed and
synthesized. The applicability of the probe for 2P microscopy
and 3D-spheroid was also assessed.
or the development of specific therapeutics to combat
cancer, cell-surface receptors (CSRs) are considered to be
Herein, we have successfully distinguished BiR positive
(HeLa)31 and negative (HEK-293)32 cells with a rational
design and synthesis of biotin appended, two-photon (2P)
active, and water-soluble fluoroprobe 5 (Scheme 1). This
particular probe contains a fluorescent propellerocein moi-
ety,22 tetraethylene glycol (TEG) as a water-soluble flexible
linker, and biotin as the specific anchor to BiR for the
endocytosis. Utilizing UV−vis., steady-state, and time-resolved
fluorescence spectroscopy, the robustness and suitability of the
probe for in vitro and in vivo applications were extensively
investigated. Further, both single photon and 2P fluorescence
microscopy were employed for 2D cell lines and 3D spheroids
for an efficient differentiation of BiR positive cells.33
Synthesis of the tricomponent probe was initiated with a
TEG linker, which was modified with propargyl bromide, and
compound 2 was obtained. Then biotin, the substrate of BiR,
was appended via a simple ester coupling to obtain 3. Finally,
to construct desired compound 5, copper(I)-assisted alkyne−
azide click chemistry was employed to tether compound 3 with
fluorescent propellerocein (Scheme 1 and Supporting In-
formation). Compound 4 was synthesized using a reported
protocol.22 Over the years, quite a few biotin-conjugated
fluoroprobes were developed for BiR targeting applications;
however, compound 5 has several advantages over them as
shown in Table S1.
F
the most important targets.1−4 These CSRs are overexpressed
in most of the cancer cells as compared to normal cells.4−7
Folate,8 insulin-like growth factor,9 epidermal growth
factor,10,11 G protein-coupled,5 and biotin receptors (BiR)12
are prototype examples of CSRs which are popular targets for
cancer theranostics.13,14 Recently, BiR has been recognized as
one of the important targets for the diagnosis and treatment of
cancer for having a strong and specific interaction with
biotin.4,15−17 Therefore, the understanding of a receptor-
mediated endocytosis pathway and identification of CSR
positive cells is crucial and prerequisite before the admin-
istration of therapeutics. Among the malignancy identification
techniques, computed tomography (CT) and magnetic
resonance imaging (MRI) were successfully used in the
medical field.18−21 Recently, fluorescence microscopy and
spectroscopy have been gaining tremendous popularity for
biomolecular sensing, trafficking, or tissue imaging because of
the benefits of high sensitivity, spatiotemporal accuracy, and
low cost.10,22,23 Organic dyes, inorganic semiconductor
quantum dot (Qdot), conjugated polymer-based nanoparticle
(Pdot), and metal nanoclusters have gained immense popular-
ity for both in vitro and in vivo applications.22,24−27 Qdot and
Pdots are shown to have superior photostability and
brightness. However, they are associated with long-term
toxicity and large size compared to the associated biomole-
cules.28,29 Recently, an intense strategic effort has been
employed for the development of small-sized organic dyes
with good water solubility, high quantum yield, photostability,
and robustness to the solution composition.22,30
It is observed quite often that synthetic modification
especially with a flexible linker may affect the photophysical
Received: August 27, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b02748
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Organic Letters
Letter
Scheme 1. Synthesis of Three-Component 2P Active
Fluorophore 5
Figure 1. Solvatochromic behavior of 5. (a) Solvent polarity-
dependent normalized fluorescence spectra and (b) fluorescence
lifetime decay profile.
Figure 3. Live-cell fluorescence imaging of BiR positive cell under
(HeLa) free-ligand competition and metabolism-block conditions.
(a−d) Cells were incubated with 5 μM probe for 1 h, and images were
acquired using phase contrast mode, blue channel (350/470
excitation/emission filter cube, nucleus stain Hoechst-33342), green
channel (490/525 excitation/emission filter cube for 5), and merge.
(e−h) Cells were treated with 1 mM of D-biotin for 1 h prior
incubation with 5. (i−l) Cells were treated with 0.05% (w/v) NaN3
for 0.5 h prior to incubation with identical concentration (image
acquired: a, e, and i phase contrast; b, f, and j blue channel; c, g, and k
green channel; and d, h, and l are merge of blue and green channel).
Table 1. Solvent-Dependent Optical Properties of 5
ab
,
b
max
max
solvent
H2O
λ
(nm)
λ
(nm)
Flu
QY
0.57
τ (ns)
abs
435
512
19.6
19.5
17.8
16.6
15.6
11.9
12.5
10.9
9.7
10.6
10.5
8.6
6.8
6.5
ethylene glycol
MeOH
EtOH
i-PrOH
MeCN
DMSO
CHCl3
EtOAc
DCM
428
425
423
421
421
426
420
423
422
422
425
416
415
418
500
495
490
485
470
469
468
467
465
463
460
458
455
452
0.71
0.58
0.54
0.56
0.38
0.50
0.48
0.30
0.45
0.37
0.32
0.28
0.25
0.24
DMF
acetone
benzene
toluene
THF
6.4
a
b
Measured with respect to 1,8-ANS in methanol.34 5% error in
measurements.
Figure 2. High photostability and robust fluorescence properties of 5
in different buffer. (a) Absorption spectra of 5 and fluorescein are
plotted after illumination under white light under identical conditions;
(b) fluorescence intensity of 5 in various commonly used buffers.
Figure 4. Imaging in 3D spheroid and using 2P fluorescence
microscopy. (a−b) 3D spheroid derived from HUVEC cells was
incubated with 10 μM of 5 and imaged under fluorescence
microscope and (c−d) live-cell fluorescence imaging under 2P (820
nm laser) excitation using HeLa cells: incubated with 5 (5 μM) and
emission collected in the range of 450−550 nm (scale 40 μm).
properties of the core fluorophore. Therefore, we wanted to
verify if there is any such undesired properties present or not.
The UV−vis. investigations show that the ground-state
behavior of 5 is hardly affected by the environmental polarity
(Figure S1, Table 1). However, in water, 5 has an emission
maximum at 512 nm whereas it shows a blue-shifted peak at
B
DOI: 10.1021/acs.orglett.8b02748
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Letter
solution and only observed minimal change in the fluorescence
intensity (Figure 2b).
After a detailed investigation of photophysical properties, we
have assayed the BiR mediated endocytosis and cellular
internalization process. In this regard, we have chosen BiR
positive cell line HeLa as the positive control14 and subjected it
to various conditions (Figure 3). In condition I, cells were
incubated with 5 μM of 5 and 1 μM of Hoechst-33342 (blue
fluorescence, nucleus-specific dye) for 1 h and imaged under a
microscope; the results are shown in Figure 3a−d. In this case,
cells showed very bright fluorescence intensity (imaged in the
green channel) due to 5, which indicated efficient cellular
internalization. However, in condition II, cells were pretreated
with 1 mM of D-biotin (substrate for BiR) for 1 h before
applying condition I. Indeed, the results shown in Figure 3e−h
clearly indicate that the cellular intake was mediated by BiR, as
the fluorescence intensity in the green channel corresponding
to 5 was very weak. The blocking of BiR is responsible for the
low level of cellular uptake of 5. To reconfirm the endocytosis
process, which is responsible for the cellular internalization, we
have utilized metabolism inhibitor NaN3 in condition III.
Similar to the biotin-block condition, the feeble fluorescence
originated due to the negligible amount of endocytosis of 5
(Figure 3i−l).
Conventionally, one-photon fluorescence microscopy is
carried out with a monolayer of cells, spread over a 2D
surface, which does not provide geometrical, mechanical, and
biochemical comparability with the real tissues. The outcome
of those in vitro studies face thoughtful criticism due to lack of
accuracy when similar experiments were conducted with the
real biological tissues.33,37 Therefore, to overcome such issues,
researchers have attempted using 3D cell culture for shrinking
the gap between real tissues and in vitro models.37 3D cell
culture models proved to be closer to the real in vivo model.
On this basis, we have tested our synthesized probe in 3D
spheroids and observed a comprehensive cellular uptake
(Figure 4a−b).
Another major problem associated with deep tissue imaging
is the penetration of the excitation light and autofluorescence.
To overcome these problems, the design of 2P active
fluorescent probes was carried out.38 The present probe
contains an ICT moiety, and generally, the molecules
containing an electron donor and acceptor groups are
established as 2P active.39,40 To validate further the
applicability of 5 for deep tissue fluorescence imaging, we
have performed 2P microscopy using an IR laser (820 nm) as
the excitation source. As per our expectation, we obtained a
strong 2P fluorescence signal with a low autofluorescence
background as shown in Figure 4c−d. After confirming the BiR
mediated endocytosis-based cellular internalization of 5 and
potentiality for use as a 2P active probe and applicability in 3D
spheroids, we extended our study toward differentiation of BiR
positive and negative cells. HeLa cells are known for the
overexpression of BiR while HEK-293 expresses an alleviated
amount compared to HeLa.14 We have cultured and incubated
them in aforementioned conditions and performed fluores-
cence microscopy. Indeed, HeLa cells showed much brighter
fluorescence as compared to HEK-293 due to better cellular
intake assisted by overexpressed BiR (Figure 5). This confirms
the differentiation between BiR positive and negative cells via
fluorescence microscopy.
Figure 5. Differentiation between BiR positive (HeLa) and negative
(HEK-293) cell lines via live-cell fluorescence imaging. (a−d) HeLa
cells were incubated with 5 for 1 h; shown are images acquired in
phase contrast, blue channel (350/470 excitation/emission filter
cube), green channel (490/525 excitation/emission filter cube), and
merge of blue and green channel, respectively. (e−h) HEK-293 cells
were incubated with 5; image sequence same as that used previously
(image acquired: a and e, phase contrast; b and f, blue channel; c and
g, green channel; and d and h are merge of blue and green channel).
452 nm in less polar solvent such as THF (Figure 1a, Table 1).
A linear correlation between the emission maxima and the
solvent polarity parameter ET(30) implies that the probe
exhibits no specific solvent effect. On the other hand, the
results from the fluorescence lifetime experiment also show
considerable solvent polarity dependence. The excited-state
lifetime became longer (Figure 1b) with the increase in solvent
polarity. The longer lifetime together with the high quantum
yield offers the possibility of fluorescence lifetime-based
imaging.
To visualize the biomolecular dynamics in live cells,
fluorescence microscopy is one of the most popular and
widely used techniques. However, photobleaching due to
exposure to light is known to affect the brightness and long-
term fluorescence imaging.35,36 To investigate the photo-
stability of 5, a comparative photostability experiment was
performed with widely used microscopy dye fluorescein (with
comparable photophysics) which revealed that 5 is far more
photochemically stable than that of fluorescein. The photo-
stability experiment was performed in PBS illuminated with a
visible lamp equipped with a photoreactor where the light
intensity was 12 000 lx. After 200 min of exposure to visible
light, fluorescein loses more than 50% of its absorbance
whereas 5 loses only up to 10% (Figure 2a). Furthermore,
fluoroprobe 5 showed remarkable thermostability up to 50 °C
(Figure S2) and pH stability over a wide range (Figure S3).
We have also monitored the fluorescence intensity of 5 in a
wide range of biologically relevant buffers and 10% SDS
In conclusion, we have successfully designed and synthesized
an indoline-based D-biotin-conjugated, 2P active, three-
C
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component, and water-soluble fluorescent dye, with strong
solvatofluorochromism. The potentiality of the probe has been
comprehensively proven by its high fluorescence quantum
yield, photostability, thermostability, and retention of
fluorescence intensity in a wide variety of buffer solutions.
After confirming BiR mediated endocytosis, we successfully
employed this molecule to differentiate between BiR positive
(HeLa) and negative (HEK-293) cells. The applicability of the
probe was also investigated with 3D spheroid and 2P
microscopy. We firmly believe that this presented strategy
with an environment-sensitive small organic dye will be highly
important for pretherapeutic diagnosis.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b02748.
Details of synthesis and cell-culture protocol, character-
ization by NMR, mass spectrometry and UV−vis
spectroscopy (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: akoner@iiserb.ac.in
ORCID
Apurba L. Koner: 0000-0002-8891-416X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the financial support from Department of
Biotechnology (DBT, BT/PR14498/NNT/28/880/2015),
India. K.P. gratefully acknowledges CSIR, India for his doctoral
fellowship. We acknowledge sophisticated instrumentation
facility IIT Indore for the two-photon imaging. We thank
Mr. Rupam Roy for his help with NMR measurements.
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